CDld proteins present alpha-galactosyl ceramides (aGCs) to NKT cells causing release of cytokines including large amounts of IFN-gamma and IL-4. These two cytokines promote Th1 and Th2 responses, respectively. Thl responses mediate the observed antitumor and antiinfective properties of aGCs. Th2 responses suppress the deleterious inflammatory effects of autoimmune processes such type I diabetes and encephalomyelitis. These two types of responses (Th1 vs. Th2) can offset one another, diminishing the effects of both. The ability to selectively stimulate Th1 or Th2 cytokine release from NKT cells via glycolipids would be an effective means of modulating the immune system. This project will explore the extent to which CDld-dependent cytokine release by NKT cells can be influenced by glycolipid structure. To determine the relationship between glycolipid structure and the CDld-mediated cytokine release profile of glycolipids, series of glycolipids will be prepared by incrementally varying the structures of each of the three portions of aGCs. Effects of length and nature of lipid chains in aGCs will be determined. The requirement for hydroxyl groups in particular stereochemical arrangements in the polar portion of ceramide will be assayed. Influences of substitution on the carbohydrates of glycolipids will be measured, including studies of the effects of groups appended at C6". The cytokine release profile caused by these compounds will be assayed using the splenocytes of transgenic mice that over express NK'I" cells. These assays will provide a correlation of glycolipid structure to cytokine production, and it is anticipated that appropriately modified aGCs will prove useful in polarizing NKT cell cytokine release resulting in either Th1 or Th2 responses. Furthermore, effects of glycolipid structure on biophysical and structural interactions with TCR and CDld will be studied in collaboration with Dr. Teyton (Project #2) and the T cell repertoire available for glycolipid recognition will be studied in mouse in collaboration with Dr. Bendelac (Project #3). Altogether, these studies will generate a comprehensive view of glycosylceramide recognition by T cells and could lead to the design of a second generation of Th1/Th2 selective aGC adjuvants with promising applications for vaccine design and the prevention and treatment of diseases.